This phase I first-in-human study was conducted in Japanese patients to investigate the safety, pharmacokinetics (PKs), and determine the maximum tolerated dose (MTD) of oral TAK-285, a novel dual erbB protein kinase inhibitor that specifically targets human epidermal growth factor receptor (EGFR) and HER2.The TAK-285 dose was escalated until MTD was determined. A second patient cohort received TAK-285 at the MTD for at least 4 weeks.In all, 26 patients received TAK-285 at doses ranging from 50 to 400 mg once daily (q.d.) or twice daily (b.i.d.); 20 patients made up the dose escalation cohort and the remaining 6 patients were the repeated administration cohort. TAK-285 was well tolerated. Dose-limiting toxicities noted in two patients who received 400 mg b.i.d. were grade 3 increases in aminotransferases and grade 3 decreased appetite. Consequently, the MTD was determined to be 300 mg b.i.d. Absorption of TAK-285 was rapid after oral dosing, and plasma exposure at steady-state increased in a dose-proportional fashion for doses ranging from 50 to 300 mg b.i.d. A partial response was observed for one patient with parotid cancer who received 300 mg b.i.d.The toxicity profile and PK properties of oral TAK-285 warrant further evaluation. Dimerisation of the human epidermal growth factor receptor (EGFR) protein family members, including HER1/EGFR and HER2, activates intracellular kinase and initiates a phosphorylation cascade that, in tumour cells, results in enhanced cellular proliferation and survival. Especially in the case of dimers that contain HER2, such activation of signal transmission can be persistent and potent, and under these circumstances is associated with high cellular differentiation and abnormal growth , and determine the maximum tolerated dose (MTD) of oral TAK-285 in patients with solid tumours. Two cohorts were planned for this study: a dose escalation cohort and a repeated administration cohort. In the dose escalation cohort, patients received a single oral dose of TAK-285, followed by 2–6 days of observation without treatment, followed by treatment with the same dose if the safety was confirmed. In this cohort, patients received TAK-285 once weekly. One cycle was 4 weeks, consisting of 3 weeks of treatment and 1 week of observation without treatment. TAK-285 was given once daily (q.d.) or twice daily (b.i.d.). The dose was escalated from a starting dose of 50 mg until the MTD was determined. In the repeated administration cohort, patients were treated with oral TAK-285 at the MTD for at least 4 weeks in order to confirm safety. Patients continued to be treated with TAK-285 at the same dose level if the treatment was well tolerated and there was no evidence of progressive disease (PD).The study was conducted in accordance with the protocol approved by the institutional review boards of the participating institutions, and with the Harmonized Tripartite Guideline of the International Conference on Harmonization for Good Clinical Practice.Patients with histologically/cytologically confirmed metastatic or advanced cancer that was unresponsive to standard therapy were eligible for this study, provided that the following criteria were met: Eastern Cooperative Oncology Group performance status of 0–1; age of 20–74 years; life expectancy of at least 12 weeks; adequate bone marrow and organ function; at least 1 measurable lesion based on Response Evaluation Criteria in Solid Tumours (RECIST) (–3) with fever of 38 °C or higher, or toxicity resulting in cessation of treatment for >21 consecutive days (including the stipulated period of observation without treatment).In the dose escalation cohort, the dose of TAK-285 was decided by consideration of adverse events (AEs) observed during the first cycle. If one of three patients had a dose-limiting toxicity (DLT), another three patients were added to the cohort. If none of three patients had a DLT, the dose in subsequent patients was increased to the next level. If there remained only one patient having a DLT, the dose was also increased to the next level, however, if >1 patient had a DLT, there was no progression to the next level. Dose escalation was continued until the MTD was determined. The dose of TAK-285 was increased by 100 or 40% in accordance with accelerated titration designs reported previously . The secondary endpoints were objective response rate (complete response (CR) and partial response (PR)), disease control rate , for at least 12 weeks), and time to tumour progression, defined as the time from the first dose of TAK-285 until disease progression or death. Tumour response was assessed every 4 weeks by RECIST version 1.0 , clinical laboratory tests, lung function tests , chest X-ray, and 12-lead electrocardiogram (ECG). These tests were performed weekly with the exception of chest X-rays (every 4 weeks), arterial blood gas analysis (at screening) and ECG . All ECG charts were submitted to the ECG evaluation committee to assess cardiac function. Adverse events were graded based on the National Cancer Institute Common Toxicity Criteria for Adverse Events, version 3.0 .–1 in plasma and 2 ng ml–1 in urine when 50 μl of the plasma sample and 55 μl of the urine sample (containing 5 μl of Tween 80 solution) were analysed. Tween 80 solution was added to the urine sample to prevent the adsorption of analytes onto the sample containers. The accuracy of the plasma assay  ranged from −10.8 to 11.0% for TAK-285, and from −14.0 to 13.8% for M-I. The accuracy of the urine assay ranged from −7.7 to 7.0% for TAK-285 and from −9.5 to 8.8% for M-I. Pharmacokinetic parameters of TAK-285 and M-I were estimated for each patient using noncompartmental methods with Phoenix WinNonlin Version 6.1 .In the dose escalation cohort, plasma samples for PK analysis were collected at predose and up to 72 h after single dose administration and on day 21 after repeated administration of TAK-285. Urine samples were also collected up to 24-h postdose. In the repeated administration cohort, plasma samples for PK analysis were collected at predose and up to 12-h postdose on days 1 and 28. Urine samples were also collected up to 12-h postdose each day. Concentrations of TAK-285 and M-I in plasma and urine were determined using validated liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. The plasma and urine samples were treated by liquid–liquid extraction and subjected to LC-MS/MS equipped with a reversed-phase column. The lower limit of quantification for both TAK-285 and M-I was 0.2 ng mlμl of preservative solution . The samples were stored at room temperature and processed within 72 h after sampling. The CellSearch System  was used for the isolation and enumeration of circulating tumour cells (CTCs), which were defined as nucleated cells lacking cluster of differentiation 45 and expressing cytokeratin.At screening and on day 15 after the start of repeated administration, 20 ml of peripheral blood was obtained and divided into two 10 ml exclusive use containers (CellSave tubes) filled with 300 A total of 26 patients were enrolled between July 2007 and May 2010, and received at least one dose of TAK-285. Demographic characteristics of patients are summarised in The 26 patients received TAK-285 at a dose ranging from 50 to 400 mg q.d. or b.i.d., 20 patients made up the dose escalation cohort and the remaining 6 patients were the repeated administration cohort. Dose-limiting toxicities observed during the study were grade 3 increased alanine aminotransferase (grade 3) and increased aspartate aminotransferase in one patient, and decreased appetite in a second patient. Both patients were treated with TAK-285 400 mg b.i.d.; hence, the MTD was determined to be 300 mg b.i.d. In the repeated administration cohort, six patients received the MTD continually for 36–169 days.Adverse events were observed in all 26 patients who received TAK-285; 22 (84.6%) of these AEs were considered to be related to TAK-285 treatment.The most common grade of AEs related to TAK-285 was grade 2 or lower in 17 patients (65.4%), grade 2 in 11 (42.3%), grade 3 in 5 (19.2%); there were no grade 4 AEs related to TAK-285 administration. As noted above, grade 3 AEs related to TAK-285 treatment included DLTs in two patients: increased alanine aminotransferase and increased aspartate aminotransferase in one patient, and decreased appetite in a second patient. These patients were withdrawn from the study because their DLTs resulted in permanent discontinuation of TAK-285 treatment. No other patients were withdrawn from the study because of TAK-285 treatment-related AEs. One of two patients with DLTs had myocardial ischaemia, which was the only serious AE related to TAK-285 treatment observed during the study. Two patients died during the study; in both cases, the cause of death was aggravation of the primary disease and was considered to be unrelated to TAK-285 treatment. The time period from last dose to death was 33 and 25 days, respectively.No clinically significant changes were observed in lung function tests, chest X-rays, vital signs or ECGs.Cmax) within 2.5 h after single oral administration. The estimated terminal-phase half-life (t1/2) was 6–9 h for TAK-285 and 7–10 h for M-I at all doses. The time to reach the maximal plasma concentration (Tmax) and t1/2 were not remarkably changed after multiple dosing. On day 21, mean plasma concentrations of TAK-285 increased over a dose range of 50–300 mg b.i.d., and mean concentrations of M-I increased over the TAK-285 dose range of 50–200 mg b.i.d. (0−tau) was suggested for TAK-285 after multiple dosing at doses ranging from 50 to 300 mg b.i.d. but was not clearly indicated for M-I (Cmax were 1.2–3.6 for TAK-285 and 1.2–2.6 for M-I. The accumulation ratios of AUC were 1.4–4.6 for TAK-285 and 1.4–3.7 for M-I. The mean cumulative excretion ratios of TAK-285 and M-I in urine (up to 12- or 24-h postdose following single or multiple dosing) were below 0.02% of the dose.The PK parameters of TAK-285 and M-I are summarised in g b.i.d. . A dose- for M-I . The accCirculating tumour cell samples both at screening and on cycle 1 day 15 after the start of repeated administration were obtained and evaluated in all patients except for those who were removed from study by cycle 1 day 15 and 1 patient with a coagulated blood sample on cycle 1 day 15. Overall, seven patients had unfavourable baseline counts (⩾5 cells per 7.5 ml blood), none of which converted to a favourable count after TAK-285 treatment. Analysis of CTC data did not show significant changes after treatment with TAK-285.One patient with parotid cancer (3.8%) achieved a PR lasting for 56 days. However, the remaining patients were reported as PD.The maximum percentage decrease from baseline in tumour size (sum of measured lesions) was evaluated separately for all patients. Only one patient in the dose escalation cohort showed reduction in tumour size, whereas three of six patients in the repeated administration cohort showed reduction in tumour size.The objective response rate and disease control rate were each 3.8% (1 of 26 patients), and the median time to tumour progression was 58 days.Clinically, TAK-285 was very well tolerated in spite of the fact that AEs were observed in all patients. The most frequent AEs were increased alanine aminotransferase and increased aspartate aminotransferase, followed by rash, increased blood bilirubin and diarrhoea; these were similar to AEs seen with other inhibitors of the EGFR family of tyrosine kinases . This event was considered to be related to TAK-285 treatment because diarrhoea that developed after the start of treatment with TAK-285 was suspected to have aggravated pre-existing ischemic heart disease. The remaining serious AEs were regarded as unrelated to TAK-285 treatment. Two patients died during the study, and the cause of death was considered to be unrelated to TAK-285 treatment for both patients.It was interesting that pneumonitis was not reported in this study and is in contrast to findings seen with gefitinib, another EGFR tyrosine kinase inhibitor . The Cmax of TAK-285 and M-I was observed up to 2.5 h after single dose administration at all doses, indicating that absorption of TAK-285 was relatively rapid after oral administration. The t1/2 values of M-I mirrored those of TAK-285 at all doses, and their concentrations in plasma declined in a similar manner. The accumulation of TAK-285 and M-I following multiple dose administration was considered to be moderate because the mean accumulation ratios for Cmax and AUC were below 4.6 for both. A dose-proportional increase in exposure to TAK-285 at steady state was indicated over the dose range tested (50–100 mg b.i.d.), but the exposure to M-I did not increase with dose at 300 mg b.i.d. This suggests that metabolism of TAK-285 to M-I by hydroxylation may be saturated at higher doses. Urinary excretion of TAK-285 and M-I was negligible and indicated that renal excretion does not contribute significantly to the clearance of either TAK-285 or M-I.In addition to evaluating the PK of TAK-285, the PK of the TAK-285 metabolite, M-I, was also evaluated. Laboratory studies revealed that M-I inhibits the kinase activities of HER2, EGFR, and HER4 with 50% inhibition concentrations of 98, 29 and 280 nmol lThe relationship between CTCs and prognosis has been reported for prostate and breast cancer (The antitumour response was rated as PR in one patient with parotid cancer in the repeated administration cohort. HER2 has been reported to be highly expressed in parotid cancer (In summary, based upon its safety, tolerability profile, PK characteristics and potential antineoplastic activity in patients with advanced solid tumours, further evaluation of TAK-285 for the treatment of patients with solid tumours appears warranted.


Molecular identification of endogenous enzymes and biologically active substances from complex biological sources remains a challenging task, and although traditional biochemical purification is sometimes regarded as outdated, it remains one of the most powerful methodologies for this purpose. While biochemical purification usually requires large amounts of starting material and many separation steps, we developed an advanced method named “proteomic correlation profiling” in our previous study. In proteomic correlation profiling, we first fractionated biological material by column chromatography, and then calculated each protein's correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward fractions. Thereafter, we could choose possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. Ultimately, this streamlined procedure requires fewer purification steps and reduces starting materials dramatically due to low required purity compared with conventional approaches. To demonstrate the generality of this approach, we have now applied an improved workflow of proteomic correlation profiling to a drug metabolizing enzyme and successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as a phosphatase of CS-0777 phosphate (CS-0777-P), a selective sphingosine 1-phosphate receptor 1 modulator with potential benefits in the treatment of autoimmune diseases including multiple sclerosis, from human kidney extract. We identified ALPL as a candidate protein only by the 200-fold purification and only from 1 g of human kidney. The identification of ALPL as CS-0777-P phosphatase was strongly supported by a recombinant protein, and contribution of the enzyme in human kidney extract was validated by immunodepletion and a specific inhibitor. This approach can be applied to any kind of enzyme class and biologically active substance; therefore, we believe that we have provided a fast and practical option by combination of traditional biochemistry and state-of-the-art proteomic technology. Molecular identification for an enzyme reaction or biologically active substance in an organism is challenging, although molecular biological methodologies such as expression cloning , recombifor example in solubilization and stability. To solve these problems, we devised a proteomic correlation profiling methodology  (Sphingosine 1-phosphate receptor 1 (S1P1)16 (SPHK2) , 20, and (SPHK2)  binds to (SPHK2) , 22. By in vitro  and fructosamine 3-kinase (FN3K)  , whereasIn this study, we have successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as the major CS-0777-P phosphatase candidate in the human kidney by proteomic correlation profiling. According to available information, this is the first report applying proteomic correlation profiling to enzyme classes other than kinases; similarly, we believe this to be first application of proteomic correlation profiling to human tissue extract, which therefore has opened up wide usage of proteomic correlation profiling for all types of enzyme identification.All chemical compounds were synthesized in Daiichi Sankyo Co., Ltd. Chemical structures are illustrated in Human frozen tissue blocks  were collected by National Disease Research Interchange, and provided by the Human Animal Bridging Research Organization. The human small intestines were provided by Xenotech. Ethical approval was obtained from the Research Ethics Committee at Daiichi Sankyo Co., Ltd.m sucrose containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.0, and protease inhibitor mixture  using a polytron homogenizer (Kinematica). The homogenate was sonicated by a Bioruptor (COSMO BIO) and filtered by a few sheets of gauze. The tissue homogenate was centrifuged with 9000 × g for 60 min at 4 °C and the supernatant was used as S9 fraction.All purification procedures were conducted at 4 °C. The block of human kidney was minced and homogenized in 9 volumes of homogenate buffer, 0.25 g for 60 min, and the precipitate equivalent to 1.1 g of human kidney was dissolved in 10 ml of homogenate buffer containing 1% n-Dodecyl-β-d-maltopyranoside (DM), and then dialyzed against 1 L of anion exchange chromatography (AEX) buffer . The dialyzed sample was filtered and loaded onto a multimodal anion exchange column . The column was equilibrated with AEX buffer over 30 min at a flow rate of 5 ml/min and the bound proteins were eluted with a linear gradient of 0–500 mm NaCl in AEX buffer for 10 min. The active fractions (10 ml) were dialyzed against 1 L of AEX buffer. The dialyzed sample was loaded onto an anion exchange column . The column was equilibrated with AEX buffer over 30 min at a flow rate of 1 ml/min. The bound proteins were eluted with a linear gradient of 0–250 mm NaCl in AEX buffer for 20 min. The active fraction (1 ml) from the Mono Q column was loaded onto a gel filtration column . The column was equilibrated with 20 mm HEPES (pH 7.0) containing 0.1% DM, 500 mm NaCl, and 1 mm dithiotreitol (DTT) over 12 h. The separation was performed by an isocratic elution with the same buffer over 30 min at a flow rate of 40 μl/min.The homogenate was centrifuged at 100,000 × m urea, 50 mm Tris-HCl, pH 8.0, 10 mm EDTA, pH 8.0, and 0.005% DM, and 10 mm DTT. The proteins were reduced at 37 °C for 20 min, followed by alkylation by incubation at 25 °C for 20 min in the dark with 20 mm iodoacetamide. The proteins were digested with 500 ng of trypsin  at 37 °C for 12 h. The reaction was stopped by acidification with 5% formic acid to a pH lower than 2.5. Samples were desalted and concentrated by using slightly modified Stage Tips  was added as the internal standard to the fractions from the gel filtration chromatography, and then the proteins were precipitated by methanol chloroform precipitation to remove the detergent and salts . Brieflyage Tips  protocol18 . Four microliters of the sample was injected to the LC-MS/MS system, and elution of peptides was carried out with a linear gradient of acetonitrile in 0.05% formic acid. Protein identification and quantitation were performed by the MaxQuant   (The LTQ-Orbitrap (Thermo Fisher Scientific) was equipped with an Agilent 1100 liquid chromatography system, which was modified to have a 200–300 nL/min flow rate by an in-house flow splitter. A homemade electrospray ionization tip column  was packed with Inertsil ODS-3 Cstitute) –35. Detam CS-0777-P or 10 μm fingolimod-P in 100 mM HEPES, pH 7.0, containing 200 μg/ml BSA, 1 mm DTT, 5 mm CaCl2, and 5 mm MgCl2. After incubation, the reaction was quenched by mixing with 2 volumes of 90% methanol/0.1% acetic acid/0.077% ammonium acetate, and 5 μl of the sample was injected in the liquid chromatography equipped with a mass spectrometry (LC-MS) system. Chromatographic separation took place on a reversed-phase chromatography column  and the analytes were analyzed with isocratic elution of methanol in 0.1% formic acid and 0.077% ammonium acetate. The flow rate employed was 0.5 ml/min and the column temperature was set at 50 °C. Eluting analytes were detected by selected ion monitoring on a single-quadrupole mass spectrometer  equipped with an electrospray ionization source and operated in positive mode. For confirmation of the inhibitory effect of an ALPL specific inhibitor on CS-0777-P and fingolimod-P phosphatase in a human kidney, the sample was incubated at 37 °C for 5 min with 1 μm CS-0777-P or 1 μm fingolimod-P in 100 mm HEPES, pH 7.4, containing 200 μg/ml BSA, 1 mm DTT, 5 mm CaCl2, and 5 mm MgCl2. After incubation, the reaction was quenched by mixing with an equal volume of 0.1% phosphoric acid/99% methanol, and 10 μl of the sample was separated by liquid chromatography followed by online tandem mass spectrometric analysis. Chromatographic separation took place on a reversed-phase chromatography column  and the analytes were analyzed with linear gradient elution of methanol in 1% formic acid and 0.077% ammonium acetate. The flow rate employed was 0.2 ml/min and the column temperature was set at 50 °C. Eluting analytes were detected by a triple quadrupole mass spectrometer  equipped with an electrospray ionization source operated in positive mode. Product ions for CS-0777, CS-0777-P, fingolimod, and fingolimod-P were detected via multiple reaction monitoring.For confirmation of the CS-0777-P phosphatase activity in human kidney samples during the purification process, in gene expression experiments of candidate proteins for CS-0777-P phosphatase and in human kidney extract after immunodepletion of ALPL, the sample was incubated at 37 °C for 30 min with 10 μm in the reaction mixture. One unit of CS-0777-P phosphatase activity was defined as the activity required for 1 ng/ml production of CS-0777 under the experimental conditions described above.Levamisole was used as an ALPL-specific inhibitor , 37, whiFollowing MaxQuant analysis, all quantified proteins within each fraction were normalized by an amount of BSA to account for any experimental error and correct for any variations in sample complexity. Each protein's normalized abundance profile was then compared against CS-0777-P phosphatase activity within each fraction while using the Pearson correlation coefficient as a measure of similarity.The total protein concentration was determined by modified Bradford protein assay  or Lowry protein assay  using BSA as a standard protein. Chromatographic fractions were assayed before spike-in of BSA internal standard for label-free quantitation.Entry clones of ALPL (clone number: FLJ93059AAAN), alkaline phosphatase, intestinal , alkaline phosphatase, placental , Lipid phosphate phosphohydrolase 1 , lipid phosphate phosphohydrolase 2 , lipid phosphate phosphohydrolase 3 , sphingosine-1-phosphate phosphatase 1 , and sphingosine-1-phosphate phosphatase 2  were obtained from the National Institute of Technology and Evaluation. Alkaline phosphatase, placental-like 2 , was obtained as a C-terminal Myc-DDK (FLAG)-tagged ORF clone with the transfection-ready DNA from ORIGENE.m HEPES, pH 7.0 containing, 0.25 m sucrose, 1 mm DTT, and protease inhibitor mixture . After sonication, the suspensions were used as an extract.ALPL and ALPP were expressed as fusion proteins with an N-terminal FLAG tag, and ALPI was expressed as a fusion protein of a C-terminal FLAG tag. ALPPL was expressed as a fusion protein with a C-terminal Myc-FLAG-tag. LPP1, LPP2, LPP3, SPP1, and SPP2 were expressed as fusion proteins with a C-terminal green fluorescence protein (GFP) tag. All proteins were expressed in human embryonic kidney 293-F cells  with 293 fectin (Invitrogen) according to the manufacturer's protocol. The transfected cells were cultured for 72 h in FreeStyle 293 Expression Medium (Invitrogen). The cells were collected by centrifugation and suspended in 10 mp-nitrophenylphosphate (data not shown).Protein expression of ALPL, ALPI, ALPP, and ALPPL was confirmed and quantified by Western blot analysis. Proteins were loaded onto SDS-PAGE gels (4–15% gradient) and then electrotransferred onto a polyvinylidene difluoride membrane by iBlot (Invitrogen). After blocking with AQUA Block , the membrane was incubated with an anti-FLAG M2 antibody conjugated with horse radish peroxidase (Sigma Aldrich), with subsequent visualization by an ECL Western blotting detection system . The band intensity was scanned by a NightOWL imaging system  and quantified using C-terminal FLAG-bacterial alkaline phosphatase (Sigma Aldrich) as a standard protein. Expressed ALPs were confirmed to have phosphatase activity by using a standard substrate of 4 cells/well in a 96-well culture plate. Bisbenzimide H 33342 trihydrochloride (Hoechst 33342) DNA dye (Sigma) was used for staining nuclei at a final concentration of 0.01 μg/ml. GFP fluorescence of these proteins and the nuclei of all cells were visualized  with ImageXpress confocal microscopy , and analyzed using MetaXpress software (Molecular Devices).Protein expression of LPP1, LPP2, LPP3, SPP1, and SPP2 was confirmed and quantified by C terminus fused GFP fluorescence. The cells transfected with these genes were plated at a density of 3 × 10DM was added to the human kidney extract at a final concentration of 0.1%, and rotated at 4 °C for 30 min. Thereafter, the extract was diluted 10-fold with TBS. Monoclonal mouse anti-ALPL antibody  or normal mouse IgG was serially diluted with PBS containing 1 mg/ml BSA. The diluted human kidney extract was incubated at 4 °C for 2 h with the diluted anti-ALPL antibody and Protein G agarose  in TBS containing 200 μg/ml BSA. Subsequently, the mixture was filtrated using a 0.45 μm spin top filter  to remove Protein G agarose and the filtrate was tested for CS-0777-P phosphatase activity. The activity was normalized by the sample diluted by the same factor without immunodepletion.in vivo are mainly responsible for the reaction. Therefore, our strategy was first to purify an enzyme from the organ with the highest enzyme activity and identify it; second, we examined the contribution of the enzyme in the organ; third, we examined the contribution of the enzyme in the whole body. We first tested CS-0777-P phosphatase activity in the human lung, kidney, liver, and small intestine to select an appropriate starting material and found equivalent activity in these tissues (data not shown). To choose one tissue among these, we then investigated the tissues of rats and monkeys, demonstrating that the kidney had the highest activity in both species (data not shown). Therefore, we decided to use the human kidney as a purification source for this study.To identify the unknown phosphatase or phosphatases capable of dephosphorylating CS-0777-P in humans, we used protein correlation profiling to identify proteins that co-purified with CS-0777-P dephosphorylation activity on fractionation. At the outset of our search, it was difficult to ascertain which organs or tissues supplemental Fig. S1, specimen #5 showed the strongest activity and thus was chosen for purification. Then, we examined biochemical characteristics for CS-0777-P phosphatase activity of the specimen, and found that; 1) the activity mainly resided in the insoluble fraction recovered after ultracentrifugation of human kidney homogenate; 2) the detergent DM effectively re-solubilized the activity; 3) activity was inactivated by EDTA, and then recovered by the addition of Ca2+, Mg2+, 4) the activity was stable from pH 5 to pH 9, 5) the observed molecular weight of the activity was 150–170 kDa as determined by gel filtration chromatography, 6) the activity was bound to a multimodal anion exchange column (Capto adhere) at pH 9 and an anion exchange column at pH 9, but was not bound to a blue dye column, hydrophobic interaction column, cation exchange column, or a hydroxyl apatite column.The schematic diagram of this study is indicated in A–3D) and we still could not locate a distinct band correlating with CS-0777-P phosphatase activity in the fractions of the final gel filtration chromatography step on SDS-PAGE gel because there were too many other bands on the gel. Thus, we applied proteomic correlation profiling to identify candidate proteins that co-purified with CS-0777-P phosphatase activity. During a proteomic correlation profiling experiment, which assumes that protein quantity correlates with protein activity, all of the samples (chromatographic fractions in this case) are subjected to proteomic analysis to quantify all proteins in parallel with a biological assay (CS-0777-P phosphatase activity). A protein that highly correlates with the desired enzyme activity suggests that the protein could be responsible for the biological activity. To quantify all proteins whose abundance profiles might correlate with CS-0777-P dephosphorylation, all fractions exhibiting target enzyme activity following gel filtration chromatography and several earlier steps of fractionation (D) were spiked with an equal amounts of BSA as the internal standard and digested by trypsin prior to LC-MS/MS analysis. Although labeling-based methods provide more accurate quantitation, they also require additional experimental steps and expensive reagent; thus, we chose label-free quantitation for its simplicity, requiring no additional experimental effort or expensive reagents. The proteins were identified and quantified by MaxQuant using an intensity-based label-free algorithm  . The highly correlated proteins were then manually evaluated based on their annotations, expression profiles, and domain structures to identify those most likely to possess the target enzyme activity. Among the top 25 most correlated proteins, ALPL was the only candidate that had a possible phosphatase domain and high correlation efficient of r = 0.9965, ranked as 2nd place among all 266 proteins  , 34, 38.proteins .i.e. ALPI, ALPP and ALPPL, to confirm whether other ALP isoforms could dephosphorylate CS-0777-P. Furthermore, because fingolimod phosphate (fingolimod-P) was reported to be dephosphorylated by LPP3 .A). In particular, ALPL, which was identified from human kidney, had 3-fold higher specific activity compared with the other three ALP isozymes (B).All four ALP isozymes showed CS-0777-P phosphatase activity A. In parisozymes A. Among isozymes B.Although recombinant LPP3 and LPP1 showed CS-0777-P phosphatase activity, we could not identify LPP3 in the purification process. Thus, to determine whether ALPL would be the major CS-0777-P phosphatase candidate in the human kidney, we examined the effects of treatment with an ALPL-specific inhibitor, levamisole , 37 and supplemental Fig. S2). We then examined the effect of levamisole on CS-0777-P phosphatase or fingolimod-P phosphatase activity from the human kidney extract using specimen #5, which was used as a purification source. We found that levamisole inhibited CS-0777-P phosphatase activity in a concentration-dependent manner, and that 1 mm of levamisole inhibited ∼80% of the activity in the extract. This indicated that ALPL, but no other ALPs or LPP proteins, was mainly responsible for the activity (A). On the other hand, 10 mm of levamisole inhibited ∼30% of fingolimod-P phosphatase activity in the extract. This demonstrated that ALPL was not responsible for the majority of fingolimod-P phosphatase activity in human kidney extract (B).To confirm the specificity of levamisole, we studied its effect on CS-0777-P phosphatase activity of recombinant ALP proteins, LPP1, and LPP3. Among these, levamisole prominently inhibited only CS-0777-P phosphatase activity of ALPL  suggested that ALPL was the major CS-0777-P phosphatase in humans.Next, to confirm the general contribution of ALPL, the effect of levamisole was examined on the other human kidney specimens. More than 50% inhibition by 1 msupplemental Fig. S4). Although the activity of ALPI and ALPPL was removed about 20% by anti-ALPL antibody, the expression of these ALP isozymes was very low in human kidney (data not shown), and thus, the decrease in CS-0777-P phosphatase activity would be due to the results of ALPL depletion from human kidney extract.As an independent confirmation, we further attempted immunodepletion experiments using an anti-human ALPL antibody. The human kidney extract was incubated with an anti-human ALPL monoclonal antibody or anti-mouse IgG as a negative control in the presence of protein G-agarose, and the immunodepleted samples were tested for CS-0777-P phosphatase activity. More than 80% of CS-0777-P phosphatase activity in the human kidney extract was immunodepleted with anti-human ALPL antibody . We confin vivo, we tested levamisole on human liver, lung and small intestine extracts or S9 fraction  . Levamisole inhibited the dephosphorylation activity in the liver and lung, suggesting that the major enzyme in those tissues would be ALPL. We also examined levamisole on the dephosphorylation of CS-0777-P in vivo using rats to detect the inhibitory effect. Unfortunately, levamisole had little influence on the blood concentration of CS-0777 in rats after administration of CS-0777-P due to the insufficient exposure of levamisole even at the maximum tolerant dose (50 mg/kg s.c.).To estimate the contribution of the ALPL Taken together, all of the data consistently support that ALPL is the major CS-0777-P phosphatase in human kidneys, whereas the contribution of other ALP proteins, LPP1 and LPP3 is low.In this study, we have successfully purified and identified the primary phosphatase candidate responsible for dephosphorylation CS-0777-P in human kidneys by improved proteomic correlation profiling, an integration of traditional biochemistry and state-of-the-art proteomics technology.m of levamisole inhibiting ∼80% of the activity in human kidney extract  we observed a single broad active peak in all purification steps ; 2) leva extract ; and 3)  extract .e.g. extensive metabolizer, poor metabolizer), and could be associated with different safety profiles and administration requirements (e.g. dosing). Indeed, the concentration of CS-0777-P, an active metabolite, in the phase I study of healthy volunteers was more than 20-fold higher than CS-0777 . Furthermore, we retrospectively applied proteomic correlation profiling in the third step fraction, anion exchange chromatography, and found that ALPL was identified as the highest ranked protein having a phosphatase domain while only one another protein had the domain. This indicated that we could have reduced one purification step further to identify ALPL ; however, there are limitations to this method. 1) If post-translational modification regulates an enzyme activity and the profile of the modified protein is different from that of the total protein, the method should not work because current proteomic correlation profiling is based on the profile of total proteins. 2) If there is an endogenous inhibitor protein in the fractions, the enzyme activity profile should not correlate with the simple protein amount of a single protein. 3) If more than one protein are responsible for the targeted enzyme activity and the profiles of those proteins are not separated in chromatography, again the enzyme activity profile should not correlate with the amount of a single protein. In addition, 4) if we cannot choose candidate proteins based on protein domain information, it is highly possible that we have too many candidates. In all cases above, adding additional purification steps and incorporating other sources of biological information could further reduce the list of candidates to a more manageable number.In proteomic correlation profiling, we first calculated each protein's correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward chromatography fractions. Then, we could select possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. By using this technology, we do not have to observe the separated bands correlating with enzyme activity; thereby, reducing the purification steps and starting materials because the required purity is much less than conventional approaches. Indeed, we could identify the ALPL as a candidate protein from only a 200-fold purification of human kidney extract in this study, although we were not able to confirm the enzyme activity solely on the basis of correlation, as there were numerous other species on the band of the SDS-PAGE gel that correlated with the enzyme activity by this 4-step purification (In this study, we enhanced the protein correlation profiling approach from our previous study by incorporating label-free intensity-based protein quantitation as implemented in the recently developed MaxQuant software suite , 34, 38.


Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 104 conidia or 217 transformants per 107 conidia were obtained under the optimized conditions when A. tumefaciens co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino A. aculeatus mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene AapksP, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in A. aculeatus. Aspergillus aculeatus no. F-50 [NBRC 108796], which was isolated from soil in our laboratory, forms black-pigmented asexual spores similar to those of Aspergillus niger. This A. aculeatus strain produces cellulases and hemicellulases that are applicable for synergistic pulp hydrolysis in combination with cellulases from Trichoderma reesei  for insertional mutagenesis; this method has been widely used as a genetic engineering technique for plant cells  to its host cells in the presence of a phenolic compound such as acetosyringone. The T-DNA is transferred as a single-stranded DNA into recipient cells by the Type IV secretion system  have long been applied for random insertional mutagenesis in fungal species . Howevernt cells  and moree oryzae , Fusariuxysporum , Colletogenarium , Cryptocoformans , Aspergiumigatus , and Asp awamori . This trn system  and predn system .A. tumefaciens is capable of transforming various fungi including the Ascomycetes, the transformation conditions must be optimized because the transformation frequencies vary among fungal species and strains. To establish an efficient AMT method for high-throughput insertional mutagenesis in A. aculeatus, we optimized the AMT conditions to effectively isolate transformants harboring single-copy T-DNA insertions at random loci. We also demonstrated that the established AMT method is applicable for functional genetic analyses.Although it has been previously demonstrated that A. tumefaciens C58C1 and the binary vector pBIG2RHPH2, which carries a hygromycin B-resistant gene between the left and right T-DNA borders, were kindly provided by Dr. Tsuji  supplemented appropriately, unless stated otherwise  and the primers pks-F_Nhe and pks-R_Nhe  to yield pAUR-PksP. The transformation of A. aculeatus was performed by the protoplast method  of 0.15 in 100 ml of induction medium (IM) with 200 μM acetosyringone (AS), 30 μg/ml of kanamycin, and 100 μg/ml rifampicin. The cells were grown at 24°C until the OD660 reached 0.2-0.8. The average numbers of A. tumefaciens cells in 100 μl of culture medium at OD660 = 0.2, 0.4, 0.6, 0.8, and 1.0 were calculated as 2.5 × 107, 5 × 107, 7.5 × 107, 1 × 108, and 1.25 × 108 cells, respectively, using a colony-counting method. In the co-cultivation on solid IM, a mixture of 100 μl of A. tumefaciens suspension and 104 A. aculeatus conidia was spread onto filter paper  on IM containing 200 μM acetosyringone (AS). After co-cultivation for 24-72 h at 24°C, the filter paper was transferred to the selection medium . When co-cultivation was performed in liquid IM, A. tumefaciens was cultured to OD660 = 0.4, harvested by centrifugation, and co-cultivated with 107 of A. aculeatus conidia in liquid IM containing 200 μM AS. After shaking at 120 rpm for 16-96 hours at 24°C, the germlings were harvested and incubated on SM.AMT was performed as described in EcoR I and Sal I or Xba I and Hind III. The EcoR I and Xba I recognition sites are located within the T-DNA region at positions 124 and 81 nt from the left and right border nick sites, respectively. The digestion of genomic DNA with EcoR I or Xba I in combination with Sal I or Hind III, for which there are no recognition sites on pBIG2RHPH2, yields relatively shorter fragments and thus helps to distinguish the fragment size. Hybridization was performed as described in hph-specific primers (HS-1com1 and HAS-2com) as a DNA probe (Table Conidia from the transformants were grown in MM containing 100 μg/ml of hygromycin B at 30°C for 50 hours on a shaker (170 rpm). Genomic DNA was isolated as described in Bgl II, EcoR I or Xba I. Bgl II sites are located outside the T-DNA region at positions 511 and 133 nt from the left and right border nick sites, respectively. Thus, digestion with these restriction enzymes produces T-DNA fragments carrying either side of the flanking sequence tag even when the T-DNA, with or without the vector backbone, is integrated into a recipient genome as concatemeric bands.A thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to obtain DNA sequences flanking the T-DNA insertions in the fungal transformants, following the methods described in Nco I, Nde I (both located in the middle of the T-DNA), EcoR I, or both Xba I and Spe I and used as a template for inverse PCR. Spe I was used to increase the possibility of obtaining fragments flanking the T-DNA because there are no Spe I recognition sites inside of the T-DNA, and this enzyme yields cohesive ends that are complementary with those produced by Xba I. Using genomic DNA digested with Nco I or Nde I as templates, the flanking sequences adjacent to the left and right borders were amplified with the primer sets HAS-4 and HAS-2com or HS-3 and HS-1com1, respectively. When genomic DNA digested with EcoR I or Xba I/Spe I was used as the template, the flanks of both sides of the borders were amplified with the primer sets HAS-4 and HS-3, respectively. The amplified DNA fragments were sequenced with the primer sets HS-4 and HAS-5.Inverse PCR was also applied to rescue the flanking sequences. Genomic DNA from each transformant was digested with Nine randomly selected transformants were cultured on MM in the absence of hygromycin B for 5 generations. Approximately 100 conidia derived from each 5th generation were spread on MM with or without 100 μg/ml of hygromycin B.A. aculeatus transformation, we first co-cultivated 1 × 104, 105, or 106 wild-type A. aculeatus conidia and an A. tumefaciens culture at OD660 = 0.8 on induction media (IM) supplemented with 200 μM of acetosyringone (AS) at 24°C for 48 hours, as described in the protocol for the AMT of Colletotrichum (4 of A. aculeatus conidia (data not shown), we further assessed the AMT conditions on IM plates with regard to the ratio of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the A. aculeatus starting material. Various concentrations of A. tumefaciens cells, at OD660 = 0.2-0.8, were co-cultivated with 1 × 104 of A. aculeatus conidia at 24°C for 24, 48, and 72 hours. The results in Table A. tumefaciens (OD660 = 1.0) tended to yield transformants with severe growth defects such as impaired hyphal elongation and conidiation. We consequently obtained a maximum transformation frequency of 30 transformants per 1 × 104 conidia, on average, when 1 × 104 conidia of A. aculeatus were mixed with 1 × 108 bacterial cells (OD660 = 0.8) and co-cultivated for 48 hours on IM plates. Protoplasts and conidia were transformed with equal efficiency by A. tumefaciens (data not shown), which enabled us to omit the intricate handling for protoplast preparation. The relatively large standard deviation in these and later experiments presumably reflects the general nature of the transformation in Aspergillus.To determine whether or not AMT is applicable for otrichum . BecauseA. aculeatus was to allow insertional mutagenesis by T-DNA insertion. To help reduce the labor requirement of the numerous media preparations or transfer of many transformants from IM to SM plates, we investigated ways in which more transformants could be obtained on an SM plate by increasing the total amount of mixed A. tumefaciens (OD660 = 0.8) and conidia spread onto an IM plate while holding the ratio of conidia to A. tumefaciens cells at the optimum value (1:104). Unexpectedly, increasing the amount of this mixture did not increase the number of transformants per plate in a dose-dependent manner because the transformation frequency was reduced , 200 μM was selected for subsequent trials because this concentration yielded the most transformants. Co-cultivation for 60 hours yielded more transformants than at 48 hours; however, more transformants with growth defects tended to emerge on the SM plates. We again investigated the effects of a higher concentration of A. tumefaciens cells and conidia in liquid IM with the same ratio of A. tumefaciens cells to conidia (50:1), but the number of transformants did not increase in a dose-dependent manner. This result may have been caused by an insufficient supply of AS for the A. tumefaciens cells to express the virulence genes because the concentration of AS was held at 200 μM. However, keeping the effective concentration of AS at the optimal level in relation to the amount of A. tumefaciens cells did not increase the number of transformants obtained (data not shown). Although the transformation frequency in liquid IM showed a 100-fold reduction compared with the solid IM, this transformation method is suitable for random insertional mutagenesis because fewer SM plates are required for the transfer of transformants from IM to SM plates. Therefore, we propose that performing AMT using liquid IM is a practical means for high-throughput insertional mutagenesis.We presumed that the failure to increase the transformant yield by increasing the total number of conidia and bacterial cells per plate was the result of the inefficient infection of the fungus by solid IM . We nextA. aculeatus isolates, we first compared the AMT frequencies of the A. aculeatus wild-type and a uridine auxotroph, the pyrG mutant. Because uridine must be added to liquid IM and SM plates to grow the pyrG mutant, we first assessed the effect of uridine addition on the AMT of the A. aculeatus wild-type  per 107 conidia per 100 ml IM was obtained at 60 h of co-cultivation with 0.2% uridine. The reduction of the transformation frequency and the long duration of the co-cultivation compared with the wild-type may be related to the reduced germination rate of the recipient conidia because the pyrG mutant never germinates or forms transformants in AMT without the addition of uridine to liquid IM (data not shown). Taking these data into account, we presumed that the germination of conidia and the physiological conditions of the recipient cells were critical for T-DNA uptake in A. aculeatus. Thus, we next investigated the AMT conditions using germinated conidia from the pyrG mutant that were pregrown for 24 h. Co-cultivation for 36 h with 0.01% uridine produced 122 transformants per 107 conidia per 100 ml of IM on average, which was 2- to 5-fold more than the amount obtained by the AMT method optimized for the wild-type strain; i.e. 48-hours co-cultivation with 0.01% uridine (24 ± 33 transformants) or 0.2% uridine added (58 ± 95 transformants). Our data demonstrate that the optimization of AMT for each isolate is necessary to establish efficient AMT methods.The transformation frequency tends to vary among different isolates of the same fungal species when AMT is performed using a method optimized for the standard strain . The trape Table . In the od Table . Using chph gene. These results revealed that the T-DNA integrated into the genomes of all transformants at random because DNA bands of various sizes were hybridized. The mitotic stability of the integrated DNA in 9 randomly selected transformants was examined after 5 rounds of mitosis on MM without hygromycin B, followed by culture on MM including hygromycin B. The average and standard deviation of the ratio of the number of colonies formed on MM with hygromycin B to that on MM without hygromycin B was 1.1 ± 0.13. The colony morphology also remained unchanged during culture. These data indicate that the integrated T-DNA is stably maintained in the recipient genome.The fates of the T-DNA in the genomes of the transformants were assessed by Southern blot analyses using genomic DNA isolated from 120 randomly selected transformants and a DNA probe that hybridizes to the 3 or 1 × 104 on solid IM . The T-DNA integration with the vector backbone was also relatively low. The AMT method optimized for liquid IM resulted in more transformants harboring the T-DNA integrated into a single locus without the vector backbone. Thus, we concluded that co-cultivation in liquid IM was suitable for the AMT of A. aculeatus.The effect of the co-cultivation conditions on the T-DNA integration pattern was investigated by Southern blot analyses. The recipient genomic DNA was isolated from transformants obtained under the following co-cultivation conditions: the ratio of bacterial cells to target conidia was 5 × 10IM Table . IntegraTo obtain the DNA sequences flanking the T-DNA inserts in the recipient genome, we adopted TAIL-PCR and inverse PCR using genomic DNA isolated from randomly selected transformants. We first performed TAIL-PCR, which produced readable segments of the recipient genome adjacent to both sides of the T-DNA from all transformants (13/13 transformants) harboring the T-DNA as a single copy. However, when the T-DNA existed in the recipient genome with the vector backbone or as concatemeric bands with or without the vector backbone at a single locus, segments derived from the vector or the T-DNA tended to be amplified rather than the recipient genome. Therefore, the DNA sequences flanking the T-DNA inserts could only be identified on one side in 5 of 13 transformants. The T-DNA integration with a vector backbone made it difficult to rescue the flanks of the T-DNA border. To rescue the recipient genome flanking the T-DNA irrespective of its integration mode, we performed inverse PCR or TAIL-PCR on the recipient genome digested with restriction enzymes, as described in the Materials and Methods section. We considered that the trimming of a vector backbone or concatemeric T-DNA fragments attached to the recipient genomic DNA would increase the rescue rate for either side flanking the T-DNA. Indeed, the flanks were obtained in 5 out of 6 transformants by TAIL-PCR and 5 out of 5 transformants by inverse PCR using recipient genomic DNA digested with restriction enzymes. However, it remains challenging to rescue the flanks if the vector backbone is attached to both sides of the T-DNA or if the selected restriction sites do not exist near the T-DNA integration locus. In this case, we confirmed that the far side of the recipient genome was effectively rescued by TAIL-PCR using circular DNA produced by digestion with a restriction enzyme followed by ligation. Furthermore, we were able to identify T-DNA flanking sequences even though the T-DNA fragments were integrated into different loci, although only in one transformant. Our data indicate that the tagged genes in almost all of the transformants could be recovered by a combination of TAIL-PCR and inverse PCR, which satisfies the requirement for a successful gene tagging protocol.Based on the above sequence analyses, we investigated how the T-DNA was inserted into the recipient genome without a large deletion of the T-DNA bordering sequence or the recipient genome sequence. Except in cases where the recipient genome was obtained with a short vector sequence, truncation of the T-DNA termini occurred in 89.5% (17/19) and 15.8% (3/19) of the border sequences on the left and right borders, respectively. Although truncations occurred with high frequency at the left terminus, the length of the truncation at the left T-DNA terminus was 8 bp on average and 42 bp at the longest. As shown in Figure A. aculeatus revealed that deletion of the recipient genome occurred in all 14 transformants analyzed, and the average length of the deletions was 1,393 bp. As shown in Figure A. aculeatus.A comparison of both sides of the flanking sequence tags adjacent to the T-DNA with the draft genome sequence of A. aculeatus alb1 and alb2, which formed colorless conidia, emerged on selective media from among approximately 11,000 transformants. Using these mutants, we assessed whether or not the established AMT method was applicable for random insertional mutagenesis. We first indentified genes disrupted by the T-DNA insertion in the alb1 mutant. A Southern blot analysis revealed that the T-DNA was inserted into a single locus in the alb1 mutant, so we performed TAIL-PCR to identify the T-DNA flanking sequences. A sequence analysis of the amplified flanks revealed that the T-DNA was inserted at 70 bp upstream of the polyketide synthase gene (the pksP gene (AapksP), Accession No. AB576490) and caused a 1,002-bp deletion in the recipient genome, which resulted in the deletion of a putative TATA box on the pksP promoter. AapksP was the only predicted gene near the T-DNA integration locus; it had 69.5% identity to the wA gene of A. nidulans (Accession no. Q03149) and 68.7% identity to the pksP gene of A. fumigatus (Accession no. EDP55264), which are involved in melanin biosynthesis and conidial pigmentation. To confirm that the deletion of the Aapksp locus resulted in the formation of the albino mutant, complementation tests were performed . Therefore, the mutation point resulting in the albino mutant corresponded to the locus for which the sequence was obtained as the T-DNA flank, thus demonstrating that AMT is a useful toolkit for gene tagging.During the process of establishing our AMT protocols, 2 albino mutants, d Figure . Transfoo Figure . FurtherA. aculeatus. This method was developed by optimizing parameters that affect the AMT frequencies such as AS concentration, the ratio of A. tumefaciens cells to A. aculeatus cells, co-cultivation conditions, and starting materials (A. aculeatus wild-type, 30 transformants per 104 conidia were formed, on average, when using solid IM for co-cultivation. The transformation frequency on solid IM was relatively higher than that for other fungi, e.g., 150-300 transformants per 106 recipients in C. lagenarium, 200 transformants per 106 recipients in A. awamori, 5 transformants per 107 recipients in A. niger, and 50 transformants per 105 recipients in N. crassa (A. tumefaciens C58C1 and a binary vector, pBIG2RHPH2, seem to be appropriate for the AMT of A. aculeatus because the transformation frequency is influenced by differences in the binary vector or bacterial strain used (The results presented here demonstrate that the developed AMT method is applicable for high-throughput insertional mutagenesis in aterials . Using t. crassa . A. tumeain used .F. oxysporum, A. awamori, and C. lagenarium, single-copy integration events are predominantly observed (A. aculeatus depends on the AMT method. A high density of A. tumefaciens increased the transformation frequency; however, undesirable integrations of the T-DNA such as multiple-copy integrations at a single locus, with or without the vector backbone, or integration at multiple loci also increased in A. aculeatus, as previously reported in Blastomyces dermatitidis (Suillus bovinus (A. tumefaciens yielded fewer transformants; however, the T-DNA predominantly existed as a single copy in the recipient genome. Although we expected to obtain more transformants by co-cultivating A. tumefaciens cells and fungal conidia in high quantities on solid or liquid IM, this technique did not yield more transformants. There is a limit to the concentration of bacterial cells and fungal conidia for efficient AMT that is likely related to the physiological conditions of A. tumefaciens and the recipient fungal cells (Another aspect that must be considered to establish efficient AMT is the integration mode of the T-DNA into the recipient genome. In observed . Howeveratitidis  and in S bovinus . In contal cells . The besA. aculeatus uridine auxotroph tended to be lower than that of the wild type with the same AMT method (Table A. awamori, average transformation frequencies of 0.2, 40, 80, and 200 transformants per 106 conidiospores were obtained (The transformation frequency for the od Table . When acobtained . The traobtained . HoweverAapksp disruptants, were discovered among 11,000 transformants. To isolate one albino mutant with a 95% probability out of 5,500 transformants from the approximately 35-Mb genome of A. aculeatus, it was calculated that the T-DNA must disrupt gene the function in a 19-kb region around the T-DNA integration locus (AapksP locus in the alb1 mutant was relatively larger than those reported for M. oryzae and Arabidopsis, in which the majority of deletions ranged from 1 to 35 bp and 11 to 100 bp, respectively (AapksP is relatively large at 6,645 bp; however, it is not large enough to increase the probability of isolating two albino mutants from 11,000 transformants. Although the T-DNA integration was initially thought to be random in Arabidopsis (Arabidopsis, the T-DNA integration occurs between the recipient genome and microhomologous sequences of the T-DNA composed of 5-bp and 2-bp sequences on the left border and right border, respectively (Arabidopsis (M. oryzae (A. aculeatus, microhomology was observed on the left border of the T-DNA; however, the other bias has not been identified thus far, although we are in the process of analyzing more transformants. A combination of T-DNA bias integration and a certain deletion length in the recipient genome can contribute to the facilitation of high-throughput insertional mutagenesis by AMT. We expect that the AMT method established here will contribute to functional genetic analyses in A. aculeatus. Moreover, we expect that the techniques described here can be applied to establish AMT techniques for other organisms.During the optimization of AMT conditions, 2 albino mutants, the on locus . The 1,0ectively . Furtherbidopsis  and yeasbidopsis , the ranectively . Furtherectively , outsidebidopsis , and in . oryzae . In A. aThe authors declare that they have no competing interests.


Materials and Methods. In this case-control study, 106 SLE patients and 149 sex, age, and ethnicity matched healthy controls were genotyped for the Fas A-670G and FasLC-844T polymorphisms by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). Results. The frequency of -670AA genotype was significantly higher in SLE patients than control group and the risk of SLE was 2.1-fold greater in subjects with AA genotype (P = 0.03). The frequency of -670A allele was significantly higher in SLE patients than in controls too . The -844CC genotype frequency was significantly higher in SLE patients than in healthy controls and the risk of SLE was 2.8-fold greater in these subjects (P = 0.01). The C allele frequency was significantly higher in patients than in controls . Increased SLE risk was observed in individuals with combined effect of Fas-670AA and FasL-844CC genotypes (P = 0.001). Conclusion. Fas-670AA and FasL-844CC genotypes were associated with SLE risk, and combined effect of -670AA and -844CC genotypes might increase SLE susceptibility.FAS/FASL pathway plays a critical role in maintaining peripheral immune tolerance; therefore, the apoptosis genes, Fas and Fas ligand (FasL), could be suitable candidate genes in human SLE susceptibility.  Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is easily confused with many disorders and can affect different organs including skin, heart, lungs, blood vessels, liver, kidneys, joints, and nervous system. This complication is characterized by malregulation of T and B cells causing production of excessive amounts of different autoantibodies and formation of immune complexes against different nuclear antigens , 2.The prevalence of SLE ranges from approximately 40 cases per 100,000 individuals among Northern Europeans to more than 200 per 100,000 persons among blacks . This diSeveral evidence indicates that abnormalities in the apoptosis (programmed cell death) process could be related to development of SLE, and the elimination of autoreactive T or B cells is impaired in this disease , 7. DelaThe importance of apoptosis in immune tolerance was distinguished on a study of the genetic defects in FAS and its ligand (FASL) in mouse models of human SLE .FAS/FAS ligand system is the main extrinsic pathway for the initiation of apoptosis in numerous cells and tissues . FAS is β. A markedly elevated basal expression of FASL is observed in subjects with the C allele compared with the T allele at nucleotide position -844  in the promoter region [FAS and FASL genes are located on chromosomes 10q24.1 and 1q23, respectively , 14. Sevr region .Several studies investigated the role of FAS and FASL gene polymorphisms in the etiology of SLE; however, the role of FAS and FASL gene polymorphisms in lupus has not been conclusively established –18. TherApproval of the study was obtained from the Ethics Committee of Zahedan University of Medical Sciences. This case-control study, performed on 106 SLE patients (9 men and 97 women) with a mean age of 31.8 ± 7.8 years, who fulfilled the 1998 American College of Rheumatology (ACR) criteria, was referred to rheumatology clinics of Ali-Ebne-Abitaleb hospital in Zahedan from 2011 to 2013. The control group consists of 149 age, sex, and ethnically matched volunteers (13 men and 139 women) with a mean age of 32.90 ± 13.3 years. The healthy controls were selected following a questionnaire survey to exclude any systemic, inflammatory, and autoimmune diseases with negative ANA test and no family relation with lupus patients. A written informed consent was obtained from all participants.Genomic DNA was extracted from ethylene diamine tetra-acetic acid (EDTA) anticoagulated peripheral blood by DNA isolation kit . FAS and FASL polymorphisms were identified by the PCR-RFLP method.The primer sequences, annealing temperature, restriction enzymes, and fragments size are shown in μL final volume which contained 20 pmol of each primer, 0.1 mmol of dNTP , 0.3 μg of genomic DNA, 1.5 mmol/L of MgCl2, 2.5 μL of 10X PCR buffer, and 1.5 unit of Taq DNA polymerase , according to the following protocol: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 1 min, annealing for 1 min at 61°C for FAS A-670G and 63°C for FASL C-844T polymorphisms; extension at 72°C for 2 min; and final extension at 72°C for 5 minutes. PCR and digested products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining.Polymerase chain reaction was performed in thermal cycler (Eppendorf-Mastercycler Gradient-Germany) in a 25 χ2 test. Comparison of quantitative variants between two groups was assessed by Student's t-test. The association between qualitative variables was analyzed using the chi-square or Fisher exact tests. Variations with P values less than 0.05 were considered significant. Odds ratio and 95% confidence interval were calculated to evaluate the strength of the associations. The statistical analysis of the data was performed using the SPSS 15.0 software. Quantitative data were presented as mean ± standard deviation. Genotypes and alleles were compared between groups by use of Clinical and demographic characteristics of SLE patients and control samples were shown in P = 0.03). The frequency of A allele was statistically higher in SLE group (58%) than in controls (49%) too ; P = 0.03). Allele and genotype frequencies of both polymorphisms were in Hardy-Weinberg equilibrium in case and control groups. The frequency of the AA genotype of FAS A-670G polymorphism was significantly higher in SLE patients than in controls, and the risk of SLE was 2.1-fold greater in individuals with AA genotype in comparison to GG genotype ; P = 0.01). The frequency of C allele was 69 and 54 percent in SLE and control groups, respectively, and was significantly different ; P = 0.001) ; = 0.001) .P = 0.03). Also 5.6-fold increased risk of SLE was found by comparing AA/TT genotype with GG/TT, GG/CT, and AG/TT genotypes between the case and control groups ; P = 0.001) ; = 0.001) . Moreover, no minor alleles frequency differences were observed between two ethnic groups and different manifestations.Established evidence shows that failure of apoptosis, or programmed cell death, is considered to contribute to the development of autoimmune disorders characterized by the impaired elimination of autoreactive T cells or B cells , 7. MoreAlthough the role of FAS-mediated apoptosis in immunity and elimination of autoreactive lymphocytes are clear, but the function of FAS or FASL as the markers of apoptosis in autoimmune disorders, especially SLE, needs more investigations . NeverthThe FAS/FASL system stimulates immune tolerance by the apoptosis induction and elimination of activated T, B lymphocytes and macrophages . Mouse model investigations indicated that FAS or FASL mutations could initiate autoimmune diseases. The essential role could be played by the FAS/FASL system in the maintenance of immune tolerance, and inhibition of autoimmune disease has been clearly established . In addition abnormal expression of FAS and FASL on T and B lymphocytes in SLE patients has been demonstrated in several reports , 27. The FAS gene has a A to G substitution in the promoter region at position -670, which decreases FAS production . Also hiSince these two polymorphisms could alter basal expression of FAS and FASL genes, recent studies have suggested that FAS and FASL gene polymorphisms may play important roles in pathogenesis of SLE.In the present study, a significant difference in AA genotype frequency was observed between SLE patients and controls in comparison to GG genotype, and the risk of SLE was 2.1-fold higher in subjects with AA genotype. Also the frequency of A allele was significantly higher than G allele in SLE patients than in healthy controls. For the first time, Wu et al. in 1996 reported an association between a 84 bp deletion within exon 4 (28 amino acid deletion) of the FASL gene and SLE in USA. This deletion in extracellular domain of the FASL gene was shown to cause defect in cell death and FASL-mediated apoptosis . Then inAn association between FAS A-670G polymorphism and development of anti-RNP antibodies was observed by Lee et al. in SLE patients . In consistent with present study, Kanemitsu reported that the A allele of FAS A-670G but not G-1377A was significantly associated with SLE .In contrast to present study Araste et al. reported no different allelic distributions at position -670 between patients and controls in an Iranian population. But they observed higher GG genotype and G allele distribution at position -1377 in SLE patients. Also they found higher soluble FAS and FAS ligand levels in the patient group and lower amounts of serum anti-SSB/La in patients with the -670GG genotype .P = 0.025). In another meta-analysis that conducted by Xiang et al., similar results were achieved [Our data supported two separate meta-analysis results that performed recently by Lee et al. in 2012 and Xiang et al. in 2013 , 33. Leeachieved , 33.Moreover, we revealed that CC genotype of FASL C-844T polymorphism was significantly higher in SLE patients than in healthy controls, and the frequency of C allele was statistically higher in SLE patients. Although several studies are performed to investigate the effect of FASL gene mutations on SLE susceptibility, to date there are only a few published reports about the association between C-844T polymorphism and SLE , 33. In 2003, Wu et al. sequenced the coding region and proximal 1 kb of promoter region of FASL gene in 14 SLE patients, 30 rheumatoid arthritis patients, and 7 normal subjects. They found an SNP at -844 within the FASL promoter region  in normal populations with an allele frequency of 0.82 for African Americans. Also they reported increased expression of FASL in individuals with -844CC genotype that could enhance autoimmunity risk. In this study a significant association between -844CC genotype and SLE was reported in the African Americans in comparison to ethnically matched healthy controls. Although they showed higher frequency of -844CC genotype in Caucasian SLE patients, the difference was not statistically significant .Similarly, in 2005, Chen et al. revealed that -844CC genotype and C allele were associated with lupus susceptibility; however, they found that the -1094A/C polymorphism is not associated with SLE . As mentioned above our findings are in line with those reported by Wu et al. and Chen et al. results and supported the association of FAS ligand C-844T polymorphism and SLE hypothesis.Since the effect of a single SNP is generally minor, it is believed that the combination effects of functionally relevant SNPs may contribute to increased SLE risk. So, we studied the combined polymorphism of FAS and FASL genes and SLE susceptibility.Our results revealed that the combination of AA/CC genotypes of FAS A-670G/FASL C-844T polymorphisms might increase SLE risk approximately 3.7-fold compared to GG/CC genotype. Also 5.6-fold increased SLE risk was observed in individuals with AA/CC genotype in comparison with GG/TT, GG/CT, and AG/TT genotypes. To the best of our knowledge, the present study is the first investigation about the synergic effect of FAS A-670G and FAS ligand C-844T polymorphisms on SLE susceptibility. These results need more study to explore gene-gene interactions and its relation to SLE susceptibility to identify individuals at increased risk of SLE and to develop preventive strategies.There were not any differences in minor allele frequencies between two ethnic groups and various SLE manifestations.In conclusion, we found an association between AA genotype of FAS A-670G polymorphism and CC genotype of FASL C-844T polymorphism with SLE risk. Also the combination of AA/CC genotypes of FAS A-670G and FASL C-844T polymorphisms indicated higher SLE risk in this Iranian population.


We report here the complete genome sequence of the porcine parvovirus (PPV) strain J-PPV, isolated from central China. Our data, together with sequence data for PPV isolates from other regions of China, will help in understanding the epidemiology and molecular characteristics of PPV field isolates in China. Parvovirus and the family Parvoviridae; it is the major causative virus in a reproductive failure syndrome in swine characterized by infertility, mummified fetuses, early embryonic death, and stillbirths (Porcine parvovirus (PPV) is a negative-oriented single-stranded DNA virus belonging to the genus llbirths . The virllbirths .Since the first occurrence of PPV infection in China was reported in 1982, several PPV strains have been isolated from many Chinese provinces , 4. To dSix pairs of oligonucleotide primers were designed according to the PPV genome sequences. The PCR products were cloned into the pMD18-T vector  and sequenced with an Applied Biosystems (ABI) 3730xl DNA analyzer. The whole genome of the J-PPV strain is 5,075 bp in length, with a G+C content of 37.91%, and consists of a 291-bp 5′ noncoding region (NCR), a 526-bp 3′ NCR, and a 4,258-bp coding region. The PPV genome includes two open reading frames (ORFs)  and 95.6% with the virulent PPV strain Kresse. The NADL-2 strain differs from the Kresse strain by 6 amino acids in the VP2 protein, which have been demonstrated to be responsible for the replication efficiency of PPV in cell culture (KF742500.The GenBank accession no. for J-PPV is


A simple two-step method was employed for preparing nano-sized gold nanoparticles-graphene composite to construct a GNPs-GR-SDS modified electrode. Hemoglobin (Hb) was successfully immobilized on the surface of a basal plane graphite (BPG) electrode through a simple dropping technique. Direct electrochemistry and electrocatalysis of the hemoglobin-modified electrode was investigated. The as-prepared composites showed an obvious promotion of the direct electro-transfer between hemoglobin and the electrode. A couple of well-defined and quasi-reversible Hb CV peaks can be observed in a phosphate buffer solution (pH 7.0). The separation of anodic and cathodic peak potentials is 81 mV, indicating a fast electron transfer reaction. The experimental results also clarified that the immobilized Hb retained its biological activity for the catalysis toward NO. The biosensor showed high sensitivity and fast response upon the addition of NO, under the conditions of pH 7.0, potential -0.82 V. The time to reach the stable-state current was less than 3 s, and the linear response range of NO was 0.72-7.92 μM, with a correlation coefficient of 0.9991. Due t in 2004 . Like ca carbon)  that adh carbon) –10.et al. [2O2. Huang et al. [2 nanocomposite could effectively facilitate the direct electron transfer of catalase to the electrode.On the other hand, due to its low-cost, large specific surface area and strong interactions with metal clusters, graphene is a promising material for catalytic applications . The diset al.  employedg et al.  also shoHowever, to date, there are few papers concerning the electrochemical response of hemoglobin on gold nanoparticles-graphene sheets composites. As a well-characterized, electron transfer protein, hemoglobin has been used extensively as a test system for direct electron transfer (DET) of redox proteins or for communication in a protein stack by assembling it with other proteins in a matrix –25.In this work, we synthesized Au nanoparticles using graphene oxide (GO) as protecting agent to give a stable red suspension. To obtain high electrical conductivity and water solubility of our GNPs-graphene composites, we added sodium dodecyl sulfate (SDS) solution to the GNPs-GO suspension prior to chemical reduction. The anionic surfactant SDS was employed as an effective solubilizing agent for graphene nanosheets and for isolating them from each other. Otherwise, the strong van der Waals interactions among the reduced graphene sheets results in their rapid aggregation in solution during the chemically reduction process. Then, water soluble GNPs-GR can be successfully obtained by the non-covalent interaction of graphene with sodium dodecylsulphate (SDS) in this procedure ,27. Furt2.2.1.Electrochemical experiments were performed with a CHI660 electrochemical analyzer  with a conventional three-electrode cell. A Hb/GNPs-GR-SDS modified BPGelectrode (Φ = 5.2 mm) was used as a working electrode. An Ag/AgCl electrode and a platinum wire electrode were used as the reference and the auxiliary electrodes, respectively.−1) was stored at a temperature of 4 °C as stock solution. Graphite, and hydrazine hydrate solution (50 wt %) were purchased from Shanghai Chemical Reagent Co. Ltd.  Saturated NO solutions were prepared as described in the previous literature [Horse heart hemoglobin was obtained from Sigma  and used without purification. Hb solution (5 mg·mLterature . In brie2.2.4 with NaBH4 in the presence of aqueous graphene oxide (GO). An aqueous solution of HAuCl4  was added into graphene oxide aqueous solution  and stirred for 5 min. After that, slow and controlled addition of NaBH4 solution  is essential to avoid gold nanoparticles from forming too fast and aggregating. This experiment was repeated with graphene oxide concentrations of 0.02, 0.11 and 0.27 mg· mL−1 as well as one experiment where no graphene oxide was added.The oxidation of the graphite was carried out based on the Hummers method . The obt2.3.SDS solution  was added to the obtained gold nanoparticles-graphene oxide suspension and stirred for 20 min. Then, hydrazine hydrate solution (20 μL) was added and stirred for 5 min. This mixture was reacted at 100 °C for 24 h. The GNPs-GO solution changed color, from red to black, after the hydrazine-vapor treatment, indicating reduction of the graphene oxide . The res2.4.−1 Hb for 3 h to form an Hb/GNPs-GR-SDS/BPG electrode. Prior to electro-chemical experiments, the electrode was rinsed thoroughly with doubly distilled water andstored at 4 ° C. The Hb/GR-SDS modified electrode without GNPs and the Hb/GNPs-SDS modified electrode without graphene were prepared in the same way, only omitting the steps of the addition of HAuCl4 solution and graphene oxide solution, respectively.Initially, the BPG electrode was polished with abrasive paper before each experiment. After a short rinse with distilled water and ultrasonic cleaning with distilled water and ethanol, the electrode was allowed to dry at room temperature. Then, the GNPs-GR-SDS suspension (10 μL) was cast on the surface of a pretreated BPG electrode with a microsyringe and the solvent (water) was allowed to evaporate at ambient temperatures. The electrode was denoted as the GNPs-GR-SDS/BPG electrode. Finally, this GNPs-GR-SDS/BPG electrode was immersed in a phosphate buffer solution (pH 7.0) containing 5 mg·mL3.3.1.−1 graphene oxide was added into the solution, the color of the solution turned a red wine color. Reducing the amount of graphene oxide gradually, the color of the solution turns red, then purple, blue, which could be explained by the aggregation of gold nanoparticles. At a low graphene concentration (0.02 mg·mL−1), the gold particles are suspended but in an aggregated state. Without the addition of any dispersant, the suspension flocculates very rapidly, which should indicate a strong agglomeration of the nanoparticles.A photograph of five GNPs-GO suspensions with different graphene oxide concentrations is shown in max from 520 to 556 nm, which is typical for increasingly larger GNP diameters. Similar red-shifts have been observed in the measured optical spectra of gold nanoparticles and are attributed to the effect of electromagnetic retardation in larger nanoparticles [articles ,31.In other words, gold nanoparticles-graphene oxide composites were successfully synthesized using graphene oxide as a dispersing agent to optimize the dispersion and the stability of the gold nanoparticles. Unlike the methods reported in previous literatures, the present method involves a facile fabrication route for the attachment of gold nanoparticles on the surface of graphene without anyorganic reagent. The resulting GNPs-GO suspension can remained as a fine dispersion at least two months. Subsequently, we added hydrazine hydrate to the GNPs-GO suspension to obtain a gold nanoparticle-graphene solution in the presence of sodium dodecyl sulfate.−1 graphene. The SEM analysis indicates that the gold nanoparticles are uniformly deposited on the graphene surface, with an average size of 50 nm. This fact showed that the presence of the oxygen functionalities at the graphene surface may play an important role on the nucleation and growth of gold nanoparticles. Furthermore, 3.2.2-saturated Hb-free phosphate buffer solution (pH 7.0). The Hb/GNPs-GR-SDS/BPG electrode gives a couple of stable and well-defined redox peaks, where anodic and cathodic peak potentials are located at −317 and −398 mV, respectively  can be estimated according to Laviron [−1. This value of Ks of our prepared modified electrodes is higher than Hb/graphene-Pt/GCE (0.14 s−1) [ Laviron , which i.14 s−1) . This in−1, less than the theoretical value of -59 mV pH−1 at 25 °C for a reversible proton-coupled single electron transfer [The effect of the pH of the supporting electrolyte on the peak potentials of the Hb was also investigated . Both retransfer ,36. Althtransfer  and Hb-Dtransfer  films:H3.3.The resulting Hb/GNPs-GR-SDS/BPG electrode exhibited a good electrocatalytic activity toward the reduction of NO. To further examine the response feature of the Hb/GNPs-GR-SDS electrode to NO, the amperometric response of the Hb/GNPs-GR-SDS modified BPG electrode to NO was recorded while successively adding NO to a continuous stirred PBS solution. In this process, a potential of −0.82 V was applied to the working electrode .−8 mol·L−1 based on 3 signal-noise ratio. The resulting response suggests that the Hb/GNPs-GR-SDS/BPG electrode can be used as a reagentless biosensor to detect NO. The determined parameters of this sensor and other modified electrodes used for the determination of NO are listed in The reductive current increases to reach a stable plateau within 3 s when NO is added to the buffer solution. This suggested that the response of the electrode to NO should be a quick responsive process. The experimental results showed that the amperometric response current is linear when the concentration of NO is in the 0.72-7.92 μM range. In addition, the detection limit of the biosensor was evaluated to be 1.2 × 103.4.The stability of the Hb/GNPs-GR-SDS electrode was investigated. Even when 400 continuous cyclic scans were carried out in the potential range from 0.1 V to −1.0 V with a 100 mV/s scan rate, no obvious change of the CV curve can be observed, suggesting that proteins can be tightly immobilized on the surface-modified BPG electrode. Eight repetitive experiments were carried out with the buffer solution containing 0.72 μM NO. The relative standard derivation for eight repetitive measurements isbelow 2.3%. The Hb/GNPs-GR-SDS modified electrode that was left to stand in pH 7.0 PBS could keep its activity for at least two weeks.4.In conclusion, a gold nanoparticle-graphene nanosheets suspension can be prepared though the chemical reduction method using SDS as protector and disperser. Hb adsorbed on the GNPs-GR-SDS modified electrode via the electrostatic interactions retains its native secondary protein structure and the direct electron transfer was achieved. The CVs of a Hb/GNPs-GR-SDS/BPG modified electrode exhibited a pair of redox peaks corresponding to the electroactive center of Hb and showed good catalytic activity for the reduction of NO. The developed approach can be potentially useful in the fields of catalysis, fuel cells, and other related fields.


Tight junctions (TJs) are mainly composed of claudins, occludin, and tight junction adhesion molecules (JAM). The invasive and metastatic phenotype of highly invasive cancer cells has been related to abnormal structure and function of TJs, and with expression of activated matrix metalloproteinases (MMPs). The relevance of these mechanisms responsible for the invasion and metastasis of ovarian carcinoma is unclear. Similarly, it is not known if the expression of claudin-6, occludin and MMP2 is related with the clinical properties of these tumors.Expression of claudin-6, occludin, and MMP2 was detected in samples of human ovarian cancer tissues by immunohistochemistry and correlated with the clinical properties of the tumors.P < 0.05). There were no differences in the expression of occludin (P > 0.05). The expression of claudin-6 and occludin in ovarian cancer was not correlated with patient age, pathological grade, clinical stage, and metastasis (P > 0.05). MMP-2 expression was enhanced with increased clinical stage and metastasis (P < 0.05), but was unrelated to patient age or tumor grade (P > 0.05). There were no apparent correlations between expression of claudin-6, occludin and MMP-2 in ovarian cancer tissue (P > 0.05).The positive expression rates of claudin-6 and MMP-2 were higher in ovarian papillary serous carcinomas than n ovarian serous adenomas  for this article can be found here:  Ovarian cancer is one of the most common neoplasms in women and epithelial tumors account for approximately 90% of ovarian malignancies . The onsThe tight junctions (TJs) are mainly located in the junctional complex of epithelial and endothelial cells, and comprise three essential membrane proteins: claudins, occludins, and tight junction adhesion molecule (JAM) . There aMMPs are a zinc ion endopeptidase enzyme family, and the main enzymes responsible for degradation of extracellular matrix and cancer invasion and metastasis ,14. TheiThis research was conducted to examine the expression of claudin-6, occludin and MMP-2 in 36 ovarian papillary serous carcinomas and 26 ovarian serous adenomas specimens, and investigate the relationship between their expression and clinical properties of the ovarian tumor patients.A total of 62 ovarian cancer patients received surgery in the first hospital of Jilin University between January 2006 and October 2007. A total of 36 ovarian papillary serous carcinoma specimens from patients ranging in age from 14 to 80 years (mean age 47 years) were included in this study. Pathological examination confirmed the presence of tumor metastasis in 13 cases but these were not identified in 23 cases. The disease stages of all the patients were classified according to TNM clinical stage (UICC 1997 staging criteria). There were 20 cases of Stage I and II ovarian cancer, 16 cases of Stage III and Stage IV cancer. Fourteen of the carcinomas were of pathological grade I, 12 cases grade II, and 10 cases of grade III. None of the cancer patients had received anticancer therapy preoperatively. Twenty-six ovarian serous adenomas served as experimental controls. The clinicopathological features of the patients are summarized in Table 2O2 for 30 min. The sections were washed with PBS three times. After a 30-min protein block with normal goat serum or normal rabbit serum, the samples were incubated with the following antibodies overnight at 4°C: anti-claudin-6 , anti-occludin , anti- MMP-2 . Immunostaining was performed using the streptavidin-biotin-peroxidase complex. The biotin-conjugated secondary antibody was incubated for 20 min at room temperature. The colour reagent diaminobenzidine  was used to visualize the bound antibody. The sections were counterstained with Mayer’s haematoxylin. The negative controls were handled in the same way but were incubated in PBS instead of primary antibody.Four-micrometer-thick tissue sections were cut from the paraffin-embedded blocks. Deparaffinization was performed using a solution containing xylene, and the sections were rehydrated with graded ethanol. The slides were placed in target retrieval solution  and boiled for 5 min in a microwave oven. After the samples had cooled for 30 min, endogenous peroxidase activity was inhibited by treatment with 3% HNegative controls were processed as above without the primary antibody or normal IgG. Ovarian cancer tissues sections with positive claudin-6, occludin and MMP-2 were stained to serve as pisitive controls. The brown staining of claudin-6, occludin and MMP-2 on the cell membrane and/or cytoplasm were as classified as positive staining. Immunostaining was observed under light microscopy with 400× magnification, and positive cells, negative cells and total cells of five different visual fields were numbered in each specimen. Scoring was performed as follows: negative (−), <5% positive tumor cells; positive (+), ≥5% positive tumor cells.P < 0.05 was considered statistically significant.All statistical analysis were performed using SPSS 12.0 software. Chi-square test and McNeamr test methods were used respectively according to the type of data to analyze whether there were significant differences or relevance between the various indicators. 2 = 7.3946, P = 0.0065). As shown in Table P = 0.6964), clinical stage (P = 0.7771), metastasis (P = 0.2674) or patient age (P = 0. 6964) in this study.Immunohistochenical staining results revealed that claudin-6 protein was localized in both the cell membrane and cytoplasm. Expression of claudin-6 was 69.4℅ (25/36) in the ovarian carcinomas and 34.6℅ (9/26) in the ovarian serous adenomas and expression of claudin-6 in ovarian papillary serous carcinomas significantly was significantly higher than that in ovarian serous adenomas , as shown in Table P = 0.9184), clinical stage (P = 0.7090), metastasis (P = 0.9231) or patient age (P = 0. 8460) . The expression of MMP-2 was positively correlated with clinical stage (P = 0.0275) (Table P = 0.0367) (Table P = 0.6820) or patient age (P = 0. 8996)  or MMP-2   (Table Correlation between the expression of claudin-6, occludin and MMP-2 was evaluated in ovarian carcinoma specimens by McNemar test. The expression of claudin-6 was not correlated with that of occludin (McNemar test χTJs located in the junctional complex of epithelial cells and endothelial cells, consist of three essential membrane proteins: claudins, occludins and JAM. These maintain cell polarity, adhesion, and regulate cell proliferation and differentiation . Recent The loss of cell polarity and abnormal permeability is a characteristic change in malignant epithelial tumors ,18. TJs Our data show that the expression of claudin-6 in 36 ovarian papillary serous carcinomas is significantly higher than in 26 ovarian serous adenomas; claudin-6 expression in metastatic tumors was significantly higher than in the tumors without metastasis. Though this difference was not statistically significant, it suggests that up-regulation of caudin-6 may play a role in the occurrence, invasion and metastasis of ovarian cancer. It is unclear why there is elevated expression of claudins and associated destruction of TJs in tumorigenesis and metastasis. However, it seems possible that the aberrant up-regulation of claudins may alter the constituent ratio of TJs. On the one hand, it may cause changes in the structure of TJs leading to loss of the integrity of TJ function, increases in cell gap and loss of robustness of the cell junction. On the other hand, it may lead to disappearance of cell polarity, weakening of intercellular adhesion and facilitation of invasion. These changes may regulate tumor cell proliferation, differentiation, survival and apoptosis though some signal transduction pathways, which play a role in occurrence, invasion and metastasis of tumor. In addition, up-regulation of claudins may activate MMPs that also promote invasion and metastasis of tumors. We speculate that up-regulation of claudin-6 may have the same mechanism play a role in occurrence, invasion and metastasis of ovarian cancer.Occludin is a transmembrane protein that has an important role in maintaining function of TJs . Our resCampo et al.  found MMOur correlation studies showed that expression of the TJ proteins claudin-6, occludin and MMP-2 were not correlated in ovarian cancer. The experimental results show that the expression of MMP-2 was up-regulated in ovarian cancer. Claudin-6 was also increased. To some extent, the high expression of MMP-2 may be due to increased Claudin-6 expression, thus speeding up the extracellular matrix degradation and resulting in the damage to the structure of TJs. Agarwal et al.  found thOur present study shows that claudin-6 and MMP-2 were both up-regulated in ovarian cancer, MMP-2 expression was enhanced with increased clinical stage and metastasis indicates that claudin-6 and MMP-2 may play an important role in the progression of ovarian cancer. However, the correlations between claudin-6 expression with occludin and MMP-2 expression have not been observed.In summary, the up-regulation of claudin-6 may result in abnormal structure and function of TJs and activation of MMP-2 both of which can result in some of the highly invasive tumor cells acquiring invasive and metastatic phenotype. Claudin-6 and MMP-2 can be used as important indicators for the judgment of malignant behavior of ovarian cancer such as invasion and metastasis. However, the exact mechanism responsible for the occurrence, progression and metastasis of ovarian cancer is not completely understood and needs further studies.The authors declare that they have no competing interests.CQ carried out part of the experiments, participated in the design of the study, performed the statistical analysis, and drafted the manuscript. LW, XJ, and DL carried out most of experiments, and helped draft the manuscript. ZL, XZ, YL, YL, MW, MY, and JL assisted with the experiments, and helped to edit the paper. All authors have read and approved the final manuscript.